PROJECT SUMMARY RNA vaccines are a growing area of interest in vaccinology. They are flexible in that the purification process should be similar from one vaccine to the other and that they can be rapidly made in response to emerging diseases. Use of these nucleic acid constructs as a vaccine platform has numerous advantages: Purification is relatively streamlined, and RNA constructs can be built in days using DNA synthesis technologies followed by transcription. This allows for rapid responses to emerging pathogen threats or pivot changes in manufacturing to adopt to new circulating strains. While these vaccines show great promise, in some cases they lack full efficacy in human trials and - like protein vaccines - may require a method of enhancing their ability to induce adaptive immune responses. We propose here preliminary studies to enable a high-throughput screen (HTS) for new adjuvant molecules for RNA vaccines. At the end of the proposed research we intend to have qualified an in vitro screen for new compound classes that can (1) enhance expression of RNA constructs; and (2) turn on NF-?B in order to enhance immunogenicity of these constructs while maintaining expression levels. The work will be performed in two Aims: First, we will Develop a Green Fluorescent Protein (GFP)-based reporter RNA screen that measures translated protein levels (Aim 1). And then we will Develop and qualify an NF-?B-based reporter screen on top of the protein expression screen from Aim 1 (Aim 2). Future studies will include deploying these assays in a high throughput setting and screening large libraries for leads that enhance expression level of RNA constructs and immunogenicity of RNA vaccines thereby becoming adjuvants for RNA vaccination.